Project Summary Addiction is a disorder of major public health concern, characterized by compulsive craving, drug seeking, and a high probability of relapse that is often spurred by the presence of drug-associated cues. Drug-induced changes in midbrain circuits, including the ventral tegmental area (VTA) and substantia nigra (SN), are thought to underlie these behaviors, but the heterogeneous mixture of neuronal subtypes and projections of the midbrain has prevented a clear understanding of the role of specific neurons and circuits in behavior. In previous studies, which utilized optogenetics methods to specifically manipulate midbrain dopamine neurons, I found a functional dissociation in the contribution of neurons projecting to the nucleus accumbens versus dorsal striatum in the motivational effects of conditioned cues, suggesting that reward processes are parcellated across anatomical divisions in the midbrain. Here, I expand on these findings to identify and compare the role of dopamine and GABA neurons in the VTA and SN in cocaine-evoked behaviors and relapse of cocaine seeking evoked by cocaine-associated cues. I will do so using state-of-the-art viral-based methods to visualize and manipulate neuronal activity. In the K99 Aims, I first propose to employ in vivo deep brain imaging to visualize the calcium dynamics of large numbers of dopamine and GABA neurons in the midbrain during cocaine exposure. Next, I propose to use chemogenetic methods, which rely on the insertion of designer receptors into target neurons, to tonically silence the activity of dopamine and GABA neurons to assess their functional role in behavioral sensitization to cocaine. These studies will define the cocaine-induced physiological responses of genetically defined VTA and SN neurons, and their role in cocaine-evoked behavior. Building on the new training and insights into the neurophysiological effects of cocaine I gain during the K99 period, I will utilize in vivo calcium imaging and optogenetics to determine to the activity patterns and temporal role of select midbrain projections in operant cocaine seeking and cue-triggered relapse during the R00 period. First, I will image activity of dopamine and GABA neurons projecting to the striatum or thalamus as animals seek cocaine and respond to cocaine-associated cues. Next, I will harness the temporal precision optogenetics to phasically manipulate these projections at discrete time points to determine their necessity and/or sufficiency for cocaine self-administration and cue-triggered relapse test. The proposed studies will provide a novel and comprehensive characterization of the circuit mechanisms by which midbrain neurons orchestrate cocaine and cocaine-cue related behaviors.